Primitive Toll-like receptor (TLR)-mediated innate immune responses trigger and shape many inflammatory and immune disorders. IL-1 induces TLR2 and TLR4 in chondrocytes and both TLRs are increased in OA cartilage in situ. Moreover, microbial TLR2 and TLR4 ligands stimulate catabolic responses in chondrocytes. Our preliminary data reveal that the shared TLR and IL-1 receptor adaptor protein MyD88 (but not soluble IL-1 receptor antagonist) blunts all inflammation-induced catabolic and differentiation responses tested in cartilage explants and cultured chondrocytes. We further observe that TLR2, TLR4 double knockout (TLR2/4 dKO) mouse cartilage explants and chondrocytes retain IL-1 responsiveness, but lose responsiveness to low molecular weight hyaluronan oligosaccharides (LMW-HA) and HMGB1, two endogenous TLR2 and TLR4 ligands present in OA joints. In addition, TLR2/4 dKO mouse chondrocytes lose the capacity to mount a catabolic response in chondrocytes with increased expression of not only hyaluronidase-2 which catalyzes LMW-HA formation but also HMGB1. The central and translational hypothesis of this proposal is that inhibition of both TLR2 and TLR4 signaling is chondroprotective both in vitro and in vivo and offers novel means to develop disease-modifying OA therapy. We will perform in vitro studies on responses of cartilage explants and chondrocytes to LMW-HA and HMGB1 to determine how TLR2 and TLR4 signaling promotes chondrocyte hypertrophy and cartilage catabolism using not only MyD88, but also TIRAP/Mal which is selectively required for TLR2 and TLR4 signaling. We will also perform in vivo studies using a surgical instability-induced knee OA model to assess if OA progression is suppressed by dual knockout of TLR2 and TLR4, and alternatively by single knockout of TIRAP/Mal, and if knockout of MyD88 has superior protective effects than knockout of IL-1 receptor for OA suppression. Completion of these studies will provide new insights into how innate immune inflammatory responses in cartilage promote OA progression and provide at least one potentially druggable target for effective OA disease modification. Relevance: Osteoarthritis (OA) is the most common joint disease related to aging. Progression of OA is promoted by low- grade inflammation in cartilage. Interleukin-1 (IL-1), an inflammatory mediator, promotes the progression of OA, but results have been incomplete for suppression of OA via IL-1 inhibition. There is a pressing need to identify mediators beyond IL-1 that drive OA progression, in order to develop true and effective disease modifying treatment of this disease. PUBLIC HEALTH RELEVANCE: Osteoarthritis (OA) is the most common joint disease related to aging. Progression of OA is promoted by low-grade inflammation in cartilage. There is a pressing need to develop effective disease modifying treatment for OA. TLR2/4 innate immune signaling in the joint represents a novel target for OA.